Penicillin G-acylase is needed in great amounts for the industrial production since it cleaves penicillin to produce 6-amino-penicillanic acid (6-APA). The 6-amino-penicillanic acid is a starting material for a broad range of semi-synthetic penicillins which in part can be synthesized with this enzyme. The enzyme is produced by microorganisms. The production of the enzyme is obtained for instance in genetic modified cells or by the induction of E. coli strains by phenyl acetic acid.
The protons which are released from penicillin G by the enzymatic cleavage can be used titrimetically for the determination of the activity. It is also possible to react the obtained 6-amino-penicillanic acid with p-dimethylamino-benzaldehyde (p-DMABA) to form a yellow coloured Schiff's base which can be determined photometrically.
Another photometric assay resides in the enzymatic cleavage of the 6-nitro-3-phenylacetamido-benzoic acid (NIPAB). The 3-amino-6-nitrobenzoic acid produced by the cleavage can be determined photometrically.
However, these known processes are less suited for the determination of low activities of penicillin G-acylase.
Therefore, several efforts have been made to find a fluorescence substrate for the penicillin G-acylase satisfying the following conditions:
(a) the cleavage product of the enzymatic reaction in regard to its fluorescence properties should be different from those of the substrate as far as possible, PA0 (b) since penicillin G-acylase has a broad activity spectrum for phenylacetic acid amides, the substrate should have a phenylacetic acid radical since this radical seems to be important for the development of an activity against the substrates; and PA0 (c) the fluorescence excitation should be in the ultraviolet range (mercury lamp) or in a range of an argon ion laser (488 nm).
Therefore, a fluorescing amine is desired which can easily be reacted with phenyl acetic acid to form a product for which the enzyme has a sufficient activity.
From U.S. Pat. No. 3,741,876 a fluorometric process for the determination of the activity of an enzyme is known wherein an enzyme and a solid substrate supported on an inert silicone matrix are reacted with each other to form a fluorescing material wherein the variation of the fluorescence with the time is measured to determine the concentration of one of both reactants. In this process dehydrogenases, oxidases, phosphatases, uricase, cholinesterase and lipase may be used as enzymes.
From EP No. 0 037 583 a process for the fluoremetric determination of the activity of fat degradating enzymes in samples containing these enzymes is known wherein the sample is combined with a substrate containing the compound which reacts with the enzyme to be tested, the substrate is excitated at a specific excitation wavelength of the corresponding fluorescing groups and the variation of the fluorescence intensity of the substrate under the action of the enzyme per time unit is measured at a specific emission wavelength of the fluorescing group, the extent of the variation being directly proportional to the enzyme activity of the sample. In this case triacyl glycerols are used as substrates which are cleaved by lipase to form free fatty acid, glycerides and glycerol.
From EP No. 0 018 112 a process for the determination of the presence of an enzyme in a biological liquid is known wherein the liquid is contacted with a synthetic chromogenic substance, the substrate containing the liquid is incubated to effect an enzymatic hydrolysis and the chromophore in the obtained hydrolyzate is determined fluorometrically. In this known process an amino acid derivative of 7-amino-4-trifluoromethyl-coumarin is used as chromogenic substrate. This process in the first place is suited for the determination of proteinase enzymes.
These known processes, too, are not suited for the qualitative and quantitative assay of penicillin G-acylase by fluorometry, in particular, at a low activity of the penicillin G-acylase.
The object of the invention was to find a chromophoric substrate satisfying the above conditions(a) to (c) and thus being suitable for the qualitative and quantitative assay of the penicillin G-acylase, in particular at a lower activity thereof.